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| United States Patent |
5,776,680
|
|
Leibowitz
, et al.
|
July 7, 1998
|
Diagnostic probes for pneumocystis carini
Abstract
The present invention pertains to a method for diagnosing for Pneumocystis
carinii by detecting the presence of a nucleic acid sequence containing
the 26S rRNA gene specific for Pneumocystis carinii. More particularly,
this invention relates to a method for diagnosing for Pneumocystis carinii
which comprises amplifying a sample of DNA from Pneumocystis carinii by
polymerase chain reaction (PCR) using species specific primers and
detecting the PCR products with species specific radioactive or
non-radioactive oligonucleotide probes. This invention also relates to a
method for diagnosing for various species of Pneumocystis carinii by
detecting the presence of a nucleic acid sequence containing the
particular 16S or 26S rRNA gene sequence specific for that species of
Pneumocystis carinii.
| Inventors:
|
Leibowitz; Michael J. (Manalapan, NJ);
Liu; Yong (Piscataway, NJ)
|
| Assignee:
|
University of Medicine & Dentistry of New Jersey (Newark, NJ)
|
| Appl. No.:
|
505509 |
| Filed:
|
July 21, 1995 |
| Current U.S. Class: |
435/6; 435/91.2; 536/23.74; 536/24.32; 536/24.33 |
| Intern'l Class: |
C12Q 001/68; C12P 019/34; C07H 021/04 |
| Field of Search: |
435/6,91.2
536/23.74,24.32,24.33
935/8,77,78
|
References Cited
| Foreign Patent Documents |
| 8803957 | Jun., 1988 | WO.
| |
| 9102092 | Feb., 1991 | WO.
| |
| 9119005 | Dec., 1991 | WO.
| |
Other References
Erlich et al, Science (1991) 252:1643-1651.
Regensburger, J. Gen Microb (1988) 134:1197-1204.
Williams BioTechniques (1989) 17:762-768.
White et al PCR Protocols: A Guide to Methods & Applications, 1990, Innis
et al, Eds, pp. 315-322.
|
Primary Examiner: Myers; Carla J.
Attorney, Agent or Firm: Muccino; Richard R.
Parent Case Text
This is a continuation application of application Ser. No. 08/298,087,
filed on 31 Aug. 1994, now abandoned, which application is a continuation
of Ser. No. 07/922,987, filed on 30 Jul. 1992, now abandoned.
Claims
We claim:
1. A method for diagnosing for Pneumocystis carinii which comprises
detecting the presence of a nucleic acid sequence containing the 26S rRNA
gene specific for Pneumocystis carinii in a sample which comprises the
steps of:
(a) treating the sample with an oligodeoxyribonucleotide polymerase chain
reaction primer for each strand of the nucleic acid sequence, four
different nucleoside triphosphates, and an agent for polymerization under
hybridizing conditions, such that for each strand an extension product of
each primer is synthesized which is sufficiently complementary to each
strand of the nucleic acid sequence being detected to hybridize therewith
and contains the 26S rRNA gene specific for Pneumocystis carinii, wherein
the primers are selected such that the extension product synthesized from
one primer, when it is separated from its complement, can serve as a
template for synthesis of the extension product of the other primer;
(b) treating the sample from step (a) under denaturing conditions to
separate the primer extension products from the templates on which they
are synthesized if the sequence to be detected is present;
(c) treating the product from step (b) with oligodeoxyribonucleotide
primers, four different nucleoside triphosphates, and an agent for
polymerization such that a primer extension product is synthesized using
each of the single strands produced in step (b) as a template, resulting
in amplification of the sequence to be detected if present;
(d) hybridizing the primer extension products from step (c) with a labeled
oligodeoxyribonucleotide probe complementary to the 26S rRNA gene specific
for Pneumocystis carinii;
(e) determining whether hybridization in step (d) has occurred to diagnose
for Pneumocystis carinii, wherein hybridization is directly proportional
to the amount of nucleic acid sequence containing the 26S rRNA gene
specific for Pneumocystis carinii present in the sampler wherein the
primers and probes are selected from the group of polynucleotides
consisting of SEQ ID NOs: 6, 7, 13, 14, 17, 19-26, and 28-30.
2. The method according to claim 1, wherein in step (d) the probe is
specific for a sequence lying between two polymerase chain reaction (PCR)
primers on the Pneumocystis carinii gene.
3. The method according to claim 1, further comprising in steps (d) and (e)
a positive control which contains the 26S rRNA gene specific for
Pneumocystis carinii and a negative control which does not contain the 26S
rRNA gene.
4. The method according to claim 1, wherein the nucleic acid sequence
containing the 26S rRNA gene specific for Pneumocystis carinii is a CDNA
copy of RNA.
5. A method for diagnosing for a species of Pneumocystis carinii which
comprises detecting the presence of a nucleic acid sequence containing the
26S rRNA gene specific for that species of Pneumocystis carinii in a
sample which comprises the steps of:
(a) treating the sample with an oligodeoxyribonucleotide polymerase chain
reaction primer for each strand of the nucleic acid sequence, four
different nucleoside triphosphates, and an agent for polymerization under
hybridizing conditions, such that for each strand an extension product of
each primer is synthesized which is sufficiently complementary to each
strand of the nucleic acid sequence being detected to hybridize therewith
and contains the 26S rRNA gene specific for that species of Pneumocystis
carinii, wherein the primers are selected such that the extension product
synthesized from one primer, when it is separated from its complement, can
serve as a template for synthesis of the extension product of the other
primer;
(b) treating the sample from step (a) under denaturing conditions to
separate the primer extension products from the templates on which they
are synthesized if the sequence to be detected is present;
(c) treating the product from step (b) with oligodeoxyribonucleotide
primers, four different nucleoside triphosphates, and an agent for
polymerization such that a primer extension product is synthesized using
each of the single strands produced in step (b) as a template, resulting
in amplification of the sequence to be detected if present;
(d) hybridizing the primer extension products from step (c) with a labeled
oligodeoxyribonucleotide probe complementary to the 26S rRNA gene specific
for that species of Pneumocystis carinii;
(e) determining whether hybridization in step (d) has occurred to diagnose
for Pneumocystis carinii, wherein hybridization is directly proportional
to the amount of nucleic acid sequence containing the 26S rRNA gene
specific for that species of Pneumocystis carinii present in the sample;
wherein the primers and probes are selected from the group of
polynucleotides consisting of SEQ ID NOs: 6, 7, 13, 14, 17, 19-26, and
28-38.
6. The method according to claim 5, wherein in step (d) the probe is
specific for a sequence lying between two polymerase chain reaction (PCR)
primers on the Pneumosystis carinii gene.
7. The method according to claim 5, further comprising in steps (d) and (e)
a positive control which contains the 26S rRNA gene specific for
Pneumocystis carinii and a negative control which does not contain the 26S
rRNA gene.
8. The method according to claim 5, wherein the nucleic acid sequence
containing the 26S rRNA gene specific for that species of Pneumocystis
carinii is a cDNA copy of RNA.
9. A method for diagnosing for a species of Pneumocystis carinii which
comprises detecting the presence of a nucleic acid sequence containing the
16S rRNA gene specific for that species of Pneumocystis carinii in a
sample which comprises the steps of:
(a) treating the sample with an oligodeoxyribonucleotide polymerase chain
reaction primer for each strand of the nucleic acid sequence, four
different nucleoside triphosphates, and an agent for polymerization under
hybridizing conditions, such that for each strand an extension product of
each primer is synthesized which is sufficiently complementary to each
strand of the nucleic acid sequence being detected to hybridize therewith
and contains the 16S rRNA gene specific for that species of Pneumocystis
carinii, wherein the primers are selected such that the extension product
synthesized from one primer, when it is separated from its complement, can
serve as a template for synthesis of the extension product of the other
primer;
(b) treating the sample from step (a) under denaturing conditions to
separate the primer extension products from the templates on which they
are synthesized if the sequence to be detected is present;
(c) treating the product from step (b) with oligodeoxyribonucleotide
primers, four different nucleoside triphosphates, and an agent for
polymerization such that a primer extension product is synthesized using
each of the single strands produced in step (b) as a template, resulting
in amplification of the sequence to be detected if present;
(d) hybridizing the primer extension products from step (c) with a labeled
oligodeoxyribonucleotide probe complementary to the 16S rRNA gene specific
for that species of Pneumocystis carinii;
(e) determining whether hybridization in step (d) has occurred to diagnose
for Pneumocystis carinii, wherein hybridization is directly proportional
to the amount of nucleic acid sequence containing the 16S rRNA gene
specific for that species of Pneumocystis carinii present in the sample;
wherein the primers and probes are selected from the group of
polynucleotides consisting of SEQ ID NOs: 4, 5, 15, 16, 18, and 27.
10. The method according to claim 9, wherein in step (d) the probe is
specific for a sequence lying between two polymerase chain reaction (PCR)
primers on the Pneumocystis carinii gene.
11. The method according to claim 9, further comprising in steps (d) and
(e) a positive control which contains the 16S rRNA gene specific for
Pneumocystis carinii and a negative control which does not contain the 16S
rRNA gene.
12. The method according to claim 9, wherein the nucleic acid sequence
containing the 16S rRNA gene specific for that species of Pneumocystis
carinii is a cDNA copy of RNA.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a method for diagnosing for Pneumocystis carinii
by detecting the presence of a nucleic acid sequence containing the 26S
rRNA gene specific for Pneumocystis carinii. More particularly, this
invention relates to a method for diagnosing for Pneumocystis carinii
which comprises amplifying a sample of DNA from Pneumocystis carinii by
polymerase chain reaction (PCR) using species specific primers and
detecting the PCR products with species specific radioactive or
non-radioactive oligonucleotide probes. This invention also relates to a
method for diagnosing for various species of Pneumocystis carinii by
detecting the presence of a nucleic acid sequence containing the
particular 16S or 26S rRNA gene sequence specific for that species of
Pneumocystis carinii.
DESCRIPTION OF THE BACKGROUND
The disclosures referred to herein to illustrate the background of the
invention and to provide additional detail with respect to its practice
are incorporated herein by reference. For convenience, the disclosures are
referenced in the following text and respectively grouped in the appended
bibliography.
Pneumocystis carinii (P. carinii) is a ubiquitous eukaryotic microorganism
causing asymptomatic infections in most humans early in childhood (1) but
causing life-threatening pneumonia in immunosuppressed hosts including
patients with Acquired Immune Deficiency Syndrome (AIDS, 2). Although
morphologically P. carinii has properties associated with both protozoa
and yeasts, the 16S rRNA coding sequence of P. carinii grown in
immunosuppressed rats most resembled that of the yeast Saccharomyces
cerevisiae (S. cerevisiae, 3). This sequence also included a 390 base pair
insertion resembling a Group I intron, located 31 nucleotides from the 3'
end of the rRNA gene (3). Absence of this sequence from mature 16S rRNA
(4) and demonstration of its ability to spontaneously excise from
transcripts of cloned fragments of the gene (5) confirmed its identity as
a self-splicing intron (6-7). The sequence of the 5S rRNA of P. carinii
grown in nude rats showed closer similarity to 5S rRNA of Amoeba and
Myxomycota than to that of Ascomycetes such as Saccharomyces (8). However,
the validity of 5S rRNA sequence analysis as a taxonomic tool has been
questioned (9). In S. cerevisiae, the 5S rRNA is encoded in the same
genomic repeated element encoding 16S, 5.8S and 26S rRNAs, but on the
opposite strand (reviewed in 10), although most eukaryotes studied do not
have the gene for 5S rRNA linked to those for the other rRNA species.
Hybridization of chromosomal DNA separated by pulsed field electrophoresis
with 16S rRNA-derived probes has localized the 16S rRNA gene of
Pneumocystis to one or two 500 kbp. chromosomal DNAS, with the gene for 5S
rRNA apparently located elsewhere (11-12).
BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A and 1B show the DNA sequence of a portion of the rRNA-encoding
gene(s) of P. carinii isolated from immunosuppressed Sprague-Dawley rats
(Sasco) and the PCR amplifications which were subsequently cloned and
sequenced. The top line represents the DNA sequence of a portion of the
rRNA-encoding gene(s) of P. carinii isolated from immunosuppressed
Sprague-Dawley rats (Sasco). The horizontal lines below represent PCR
amplifications which were subsequently cloned and sequenced. Thin lines
(FIG. 1A) refer to PCR products from Sprague-Dawley rats (Sasco) and heavy
lines (FIG. 1B) refer to PCR products from Hooded rats. Numbers refer to
oligonucleotide primers (Table 1) used in each PCR reaction.
FIG. 2 shows the total contiguous sequence determined for P. carinii from
immunosuppressed Sprague-Dawley rats (Sasco) by the strategy shown in FIG.
1A. Except for the last 18 nucleotides (shown in lower case), capital
letters indicate rRNA coding sequences (positive strand), lower case
letters indicate spacers, and underlined lower case letters indicate Group
I introns. The initial 22 nucleotides are from the 3'-terminal portion of
the Group I intron in 16S rRNA. Nucleotides 23-53 are the second exon of
16S rRNA, 54-216 are internal transcribed spacer 1 (ITS1), 217-374 the
gene for 5.8S rRNA (identified by similarity to other 5.8S rRNA
sequences), 375-556 ITS2, and 557-4256 are the gene for 26S rRNA, with a
Group I intron sequence in lower case underlined. This sequence has been
deposited at EMBL/GenBank under accession No. M86760.
FIG. 3 shows a comparison of the sequence of the 5.8S rRNA gene of P.
carinii shown in FIG. 2 with the homologous sequences from Saccharomyces
cerevisiae (23) shown as Sc, Tetrahymena pyriformis (T. pyriformis) (24)
shown as Tp, and Homo sapiens (25) shown as Hs. Since the actual 5.8S rRNA
sequence was not determined, the termini of the P. carinii gene have been
chosen based on the known sequence of the homologous gene of S.
cerevisiae, to which it appears to be closely related. The three
nucleotides 5' to the proposed rRNA 5' terminus are shown here in lower
case letters.
FIG. 4 is a dendrogram generated by the "pileup" program of the Wisconsin-
GCG package indicating sequence similarity (but not necessarily
evolutionary relationships) among the 5.8S rRNAs compared in Table II.
FIG. 5 shows a comparison of the sequence of the 26S rRNA genes of P.
carinii (Pc) from FIG. 2, with homologous sequences from S. cerevisiae
(Sc), and T. pyriformis (Tp). The Group I self-splicing introns in the P.
carinii and T. pyriformis genes have been omitted. The final 18
nucleotides of the P. carinii sequence were determined from organisms from
immunosuppressed Hooded rats as shown in FIG. 2.
FIG. 6A shows the secondary structure into which the apparent Group I
intron in the gene for 26S rRNA of P. carinii can be folded. The helices
P1-P9 are conserved among Group I introns (6-7). The bases in the intron
are numbered 1 through 355, and the flanking exon regions are shown in
lower case letters. The consensus sequences P (nucleotides 80-91), Q
(nucleotides 202-211), R (nucleotides 247-260) and S (nucleotides 316-327)
are shown in boldface. FIG. 6B shows an alternative folding for the P8
helix of the intron (5) in the 16S rRNA gene.
FIG. 7 shows the sequence of the region from nucleotides 485 through 964 of
the 26S rRNA gene from P. carinii from Sprague-Dawley rats, as shown in
FIG. 5 (Pcd). This sequence was determined for three PCR products made
using oligonucleotides 4016 and 2892 as primers and for PCR products made
using the oligonucleotide pair 3425 and 3426, and the pair 2893 and 2982,
each resulting in products partially overlapping this region. This entire
sequence was thus determined on four or five isolates, with four separate
sequence determinations made for each PCR product. The sequence of DNA
amplified using the same primers (4016 and 2892) from P. carinii from
Hooded rats is shown as Pc2. The homologous regions of genes from S.
cerevisiae (Sc) and T. pyriformis (Tp) are also shown. The numbering is
according to the 26S rRNA sequence of Pcd as in FIG. 5. The sequence
denoted Pc2 has been deposited at EMBL/GenBank under accession No. 86761.
FIG. 8 shows a comparison of the sequences of the region from nucleotides
2911 through 3327 of the 26S rRNA gene of P. carinii (Pcd) from
Sprague-Dawley rats (FIG. 5) with the homologous regions from P. carinii
from Hooded rats (Pc2) and from S. cerevisiae (Sc) and T. pyriformis (Tp).
The fragment denoted Pc1 was amplified using primers 4138 and 4170. The
sequence shown for Pc2 was determined based on amplifications using primer
pair 4138 and 4139 and pair 4169 and 4170, and ligation-dependent PCR
amplification of a fragment extending from oligonucleotide 3427 through a
PstI site 381 nucleotides past the 3' end of the 26S rRNA gene. The
sequences of homologous regions of the 26S rRNA genes of S. cerevisiae
(Sc) and T. pyriformis (Tp) are shown.
FIG. 9 shows the results of PCR amplification confirming the sequence
differences between Pc1 and Pc2 shown in FIGS. 8 and 10. Primers 4358 and
4746 were used to amplify Pcd (lane 1) or Pc2 (lane2) DNA templates.
Primers 4743 and 4744 were used to amplify Pc1 (lane 3) or Pc2 (lane 4)
DNA. Lanes N contain a mixture of HindIII digested bacteriophage lambda
DNA and HaeIII digested replicative form DNA of bacteriophage phiXl74
(BRL).
FIG. 10 shows the sequence of the spacer region 3' to the 26S rRNA gene of
P. carinii from Hooded rats (FIG. 10), which was determined by
ligation-dependent PCR as described in the text. The sequences shown in
FIGS. 8 and 10 have been deposited at EMBL/GenBank under accession No.
M86759.
SUMMARY OF THE INVENTION
The present invention pertains to a method for diagnosing for Pneumocystis
carinii which comprises detecting the presence of a nucleic acid sequence
containing the 26S rRNA gene specific for Pneumocystis carinii in a sample
which comprises the steps of:
(a) treating the sample with an oligodeoxyribonucleotide primer for each
strand of the nucleic acid sequence, four different nucleoside
triphosphates, and an agent for polymerization under hybridizing
conditions, such that for each strand an extension product of each primer
is synthesized which is sufficiently complementary to each strand of the
nucleic acid sequence being detected to hybridize therewith and contains
the 26S rRNA gene specific for Pneumocystis carinii, wherein the primers
are selected such that the extension product synthesized from one primer,
when it is separated from its complement, can serve as a template for
synthesis of the extension product of the other primer;
(b) treating the sample from step (o) under denaturing conditions to
separate the primer extension products from the templates on which they
are synthesized if the sequence to be detected is present;
(c) treating the product from step (b) with oligodeoxyribonucleotide
primers, four different nucleoside triphosphates, and an agent for
polymerization such that a primer extension product is synthesized using
each of the single strands produced in step (b) as a template, resulting
in amplification of the sequence to be detected if present;
(d) hybridizing the primer extension products from step (c) with a labeled
oligodeoxyribonucleotide probe complementary to the 26S rRNA gene specific
for Pneumocystis carinii;
(e) determining whether hybridization in step (d) has occurred.
In another embodiment, the present invention pertains to a method for
diagnosing for a species of Pneumocystis carinii which comprises detecting
the presence of a nucleic acid sequence containing the 26S rRNA gene
specific for that species of Pneumocystis carinii in a sample which
comprises the steps of:
(a) treating the sample with an oligodeoxyribonucleotide primer for each
strand of the nucleic acid sequence, four different nucleoside
triphosphates, and an agent for polymerization under hybridizing
conditions, such that for each strand an extension product of each primer
is synthesized which is sufficiently complementary to each strand of the
nucleic acid sequence being detected to hybridize therewith and contains
the 26S rRNA gene specific for that species of Pneumocystis carinii,
wherein the primers are selected such that the extension product
synthesized from one primer, when it is separated from its complement, can
serve as a template for synthesis of the extension product of the other
primer;
(b) treating the sample from step (a) under denaturing conditions to
separate the primer extension products from the templates on which they
are synthesized if the sequence to be detected is present;
(c) treating the product from step (b) with oligodeoxyribonucleotide
primers, four different nucleoside triphosphates, and an agent for
polymerization such that a primer extension product is synthesized using
each of the single strands produced in step (b) as a template, resulting
in amplification of the sequence to be detected if present;
(d) hybridizing the primer extension products from step (c) with a labeled
oligodeoxyribonucleotide probe complementary to the 26S rRNA gene specific
for that species of Pneumocystis carinii;
(e) determining whether hybridization in step (d) has occurred.
In yet another embodiment, the present invention pertains to a method for
diagnosing for a species of Pneumocystis carinii which comprises detecting
the presence of a nucleic acid sequence containing the 16S rRNA gene
specific for that species of Pneumocystis carinii in a sample which
comprises the steps of:
(a) treating the sample with an oligodeoxyribonucleotide primer for each
strand of the nucleic acid sequence, four different nucleoside
triphosphates, and an agent for polymerization under hybridizing
conditions, such that for each strand an extension product of each primer
is synthesized which is sufficiently complementary to each strand of the
nucleic acid sequence being detected to hybridize therewith and contains
the 16S rRNA gene specific for that species of Pneumocystis carinii,
wherein the primers are selected such that the extension product
synthesized from one primer, when it is separated from its complement, can
serve as a template for synthesis of the extension product of the other
primer;
(b) treating the sample from step (a) under denaturing conditions to
separate the primer extension products from the templates on which they
are synthesized if the sequence to be detected is present;
(c) treating the product from step (b) with oligodeoxyribonucleotide
primers, four different nucleoside triphosphates, and an agent for
polymerization such that a primer extension product is synthesized using
each of the single strands produced in step (b) as a template, resulting
in amplification of the sequence to be detected if present;
(d) hybridizing the primer extension products from step (c) with a labeled
oligodeoxyribonucleotide probe complementary to the 16S rRNA gene specific
for that species of Pneumocystis carinii;
(e) determining whether hybridization in step (d) has occurred.
DETAILED DESCRIPTION OF THE INVENTION
This invention relates to a method for diagnosing for Pneumocystis carinii
by detecting the presence of a nucleic acid sequence containing the 26S
rRNA gene specific for Pneumocystis carinii. More particularly, this
invention relates to a method for diagnosing for Pneumocystis carinii
which comprises amplifying a sample of DNA from Pneumocystis carinii by
polymerase chain reaction (PCR) using species specific primers and
detecting the PCR products with species specific radioactive or
non-radioactive oligonucleotide probes. This invention also relates to a
method for diagnosing for various species of Pneumocystis carinii by
detecting the presence of a nucleic acid sequence containing the
particular 16S or 26S rRNA gene sequence specific for that species of
Pneumocystis carinii.
The term "oligonucleotide" as used herein refers to primers, probes,
oligomer fragments to be detected, oligomer controls, and unlabeled
blocking oligomers. Oligonucleotide are molecules comprised of two or more
deoxyribonucleotides or ribonucleotides.
The term "primer" as used herein refers to an oligonucleotide, preferably
an oligodeoxyribonucleotide, either naturally occurring such as a purified
restriction digest or synthetically produced, which is capable of acting
as a point of initiation of synthesis when subjected to conditions in
which synthesis of a primer extension product, which is complementary to a
nucleic acid strand, is induced, i.e., in the presence of nucleotides, an
agent for polymerization such as a DNA polymerase, and a suitable
temperature and pH. The primer must be sufficiently long to prime the
synthesis of extension products in the presence of the polymerization
agent.
In accord with the method of the present invention, the sequence of the
portion of the major rRNA-encoding operon (encoding the 16S, 5.8S and 26S
rRNA molecules specific for P. carinii) from organisms derived from the
lungs of immunosuppressed rats, including the genes for 5.8S and 26S
rRNAs, has been determined. These two genes show similarity to the
homologous genes of S. cerevisiae, with the gene for 26S rRNA also
containing an apparent Group I self-splicing intron.
The relatedness of different Pneumocystis isolates has been difficult to
determine in the absence of a long-term culture method for this organism.
The 5S rRNA gene amplified by polymerase chain reaction (PCR) from
multiple infected humans and rats had the identical sequences (13).
However, rat and human-derived organisms showed sequence differences in
their mitochondrial DNA (14). When portions of the 26S rRNA gene from two
different sources were sequenced, phylogenetically variable regions of the
gene were found to be different between these two organisms. This marked
sequence difference between 26S rRNA gene sequences may represent
differences between clones of the same species or may indicate the
existence of more than one species within the genus Pneumocystis. In
either case, such differences may provide a mechanism of recognizing the
relationships between different individual Pneumocystis isolates for
epidemiological studies. This appears to be the first such difference
reported between Pneumocystis isolates in the sequence of a chromosomal
gene.
The rRNA Operon of P. carinii
Although the exact phylogenetic relationship of P. carinii to other species
remains unknown, the 5.8S and 26S rRNA genes, like that for 16S rRNA (3),
are similar in primary sequence to the homologous genes of S. cerevisiae.
This finding contrasts with the report that the 5S rRNA gene most
resembles the sequence of the homologous genes of Amoeba or Myxomycota
rather than those of the Ascomycetes (8). The organization of the major
rRNA operon of P. carinii differs from that of S. cerevisiae in that for
the former there is no evidence that the 5S rRNA and 16S-5.8S-26S rRNA
operon genes are part of the same repeated DNA unit, based on pulsed field
electrophoresis studies (11-12). Linkage of the 5S rRNA gene to genes
encoding 16S rRNA or 26S rRNA by PCR techniques has not been observed. The
amount of DNA obtained from P. carinii was limited, and so classical
Southern analysis was not attempted.
The presence of Group I self-splicing introns in the 16S and 26S rRNA genes
of P. carinii distinguishes this organism from S. cerevisiae and from its
mammalian hosts. Since various compounds can specifically inhibit the
splicing of Group I introns in vitro (31), Group I intron splicing may
provide a specific target for development of new therapeutic agents
against P. carinii.
Taxonomy of P. carinii
The exact taxonomic relationships of P. carinii remain uncertain, in part
due to the limited number of eukaryotic microorganisms whose rRNA
sequences are known. Furthermore, the definitions of the groups denoted as
Fungi and Protozoa are so broad and imprecise that each includes very
distantly related organisms. It is possible that once more organisms of
this type are studied, these two groupings may prove to be inadequate, and
the taxonomy of the eukaryotic microorganisms may require some
redefinition. This has already proven to be the case for the
aicrosporidia, which have been placed in a group distinct from all other
eukaryotic microorganisms on the basis of their rRNA sequences (32).
In the absence of a long-term culture method or other tools for comparison
of different P. carinii organisms, the number of species within the genus
Pneumocystis is undefined. Antigenic differences between P. carinii
obtained from different mammalian host species have been demonstrated
(33-36), although their genetic basis is not proven. Although the 5S rRNA
gene sequences of multiple human and rat isolates of P. carinii are
identical (13), such isolates differ in the sequence of their
mitochondrial DNA (14). DNA hybridization methods with a cloned DNA
fragment have also suggested the non-identity of human and rat-derived P.
carinii, with differences noted among different human, but not rat,
isolates (37). Based on these results, it has been suggested that
subspecies of P. carinii may be designated based on the hosts from which
they are isolated (38).
The data presented herein show that multiple differences exist between the
26S rRNA gene sequences of P. carinii from Sprague-Dawley rats from Sasco
which were immunosuppressed in isolation (and therefore presumably
infected at some other location prior to their arrival here) and Hooded
rats which were immunosuppressed here without isolation (and therefore
presumably infected in this building or at some geographic location
distinct from the site at which the Sprague-Dawley rats were infected).
Since multiple independent PCR amplifications of portions of the 26S rRNA
gene prepared from templates derived from different individual rats of the
same type yielded identical sequences, there is no evidence that the
differences observed between the two sources represent PCR artefacts,
sequencing errors, or heterogeneity of rRNA sequences within an individual
cell, as has been reported in Plasmodium species (39). This variation
between different P. carinii isolates resembles that seen between
different individual humans, which also occurs in regions of the 26S rRNA
gene which are phylogenetically non-conserved (40). Sequence differences
in rRNA genes have been suggested as defining species differences within
the genus Giardia (41).
When Pc1 DNA template was amplified by PCR using the primer pair 4358
(universal) and 4746 (Pc1-specific), the expected 2,067 bp product was
produced; in contrast, no product was generated from Pc2 template with
these same primers (FIG. 9). Similarly, primers 4743 (Pc2-specific) and
4744 (Pc2-specific) amplified an approximately 3.0 kbp product from Pc2
template; no similar product was seen with Pc1 template (FIG. 9). Note
that in some reactions a barely detectable band of the same size seen with
Pc2 template was seen with Pc1 template using the latter primer pair.
These data are consistent with Pc1 and Pc2 each containing predominantly
genes encoding single distinct major 26S rRNA sequences.
Comparisons of the sequences of multiple P. carinii rRNA gene regions
should determine the extent of variability present. If different human
isolates of this organism vary as much as do different rat isolates, then
these sequences could be useful as epidemiological markers for identifying
strains of P. carinii and studying the spread of the organism and the
relative roles of new infection versus reactivation of earlier
asymptomatic colonization in the development of P. carinii pneumonitis in
immunosuppressed humans, including patients with AIDS. Since different
species of Tetrahymena differ more in their intron sequences than in the
sequences of adjacent conserved regions encoding rRNA (27), such regions
may prove to be even more variable between different P. carinii organisms.
Further studies may determine the variability within and between species
of the internal transcribed spacers (between the 16S and 5.8S rRNA and
5.8S and 26S rRNA genes) and external transcribed spacers (flanking the
rRNA coding regions). If these spacers contain regions with specific
functions in rRNA transcription or processing (30), such regions may show
sequence conservation.
The present invention is further illustrated by the following examples
which are not intended to limit the effective scope of the claims. All
parts and percentages in the examples and throughout the specification and
claims are by weight of the final composition unless otherwise specified.
EXAMPLES
METHODS
Growth and Purification of Pneumocystis carinii Sprague-Dawley rats from
Sasco, Inc. (Omaha, Neb.) were maintained in isolation cages with
protective filters (Lab Products, Maplewood, N.J.) with immunosuppression
by addition of dexamethasone (1 mg/ml) and tetracycline (0.5 mg/ml) to
their drinking water. Water and autoclaved 8% protein diet (ICN) were
provided ad libitum. Hooded rats (Harlan-Sprague-Dawley, Indianapolis,
Ind.), were treated in the same way but not isolated. Rats were sacrificed
after 8-12 weeks of immunosuppression or when signs of respiratory
distress were observed. All subsequent procedures were done at 4.degree.
C. Each pair of lungs was removed, minced with a scissors and the
homogenate was suspended in 25 ml of Dulbecco's Modified Eagle's Medium
(DMEM) and centrifuged for 10 minutes at 200.times.g to remove tissue
debris and lung cells. The supernatant was then transferred to a fresh
tube, cells were collected at 1,600.times.g and resuspended in 3 ml of
phosphate buffered saline (PBS). Suspended cells were loaded on
discontinuous Percoll gradients (10-40% in 10% steps) and after
centrifugation at 1,600.times.g for 30 minutes, trophozoites were found at
the 10-20% interface, cysts with some trophozoites and a few mammalian
cells at the 20-30% interface, and predominantly mammalian cells with some
cysts at the 30-40% interface.
For in vitro cultivation of P. carinii, mink lung cells of line ATCC CCL64
(15) grown to 80% confluence in 10 cm petri dishes in DMEM supplemented
with 10% fetal calf serum were used as feeder cells. Percoll gradient
purified cysts (5.times.10.sup.5) were added to each plate in the presence
of penicillin, streptomycin, gentamicin and fungizone, followed by
incubation at 37.degree. C. in a humidified 5% CO.sub.2 incubator. After
1-3 days in culture, the plates were gently agitated and the
Pneumocystis-containing medium was collected and centrifuged at
100.times.g for 5 minutes to pellet contaminating detached mammalian
cells. Only a few mammalian cells detached during the culture period and
these were efficiently removed by the centrifugation.
Microscopic Techniques
Pneumocystis trophozoites were quantitated in 5 ul samples air dried on
microscope slides and stained with Diff-Quik (Baxter Healthcare Co.,
Miami, Fla.). Cysts were identified by toluidine blue 0 stain (16). All
quantitation was done by counting three 5 ul samples for a total of 30 oil
immersion fields for each sample. All cultures and purified Pneumocystis
preparations were negative for fungal and bacterial contamination by
microscopy and culture, and for Mycoplasma contamination by MycoTect kit
(Gibco BRL).
Extraction of Nucleic Acids from Trophozoites
P. carinii cells from mink lung cell cultures were harvested by
centrifugation at 3,000 rpm for 30 minutes at 4.degree. C. in a Sorvall
SS-34 rotor, and were washed with chilled PBS. Cells were resuspended in
50 mM Tris-HCl ›Tris (hydroxymethyl) aminomethane hydrochloride!, 50 mM
Na-EDTA (sodium ethylenediaminetetraacetic acid), pH 8.0, and were lysed
by in cubation at 65.degree. C. for 30 minutes in the presence of 1% SDS
(sodium dodecyl sulfate). Proteins were removed by precipitation on ice in
the presence of 1.25N potassium acetate followed by centrifugation at room
temperature. Total nucleic acids were then concentrated by precipitation
in an equal volume of absolute ethanol on ice.
Oligonucleotides
DNA oligonucleotides were synthesized by beta-cyanoethyl phosphoramidite
chemistry on automated DNA synthesizers (Cyclone, Milligen and 380B,
Applied Biosystems), and were purified by chromatography on NENsorb-Prep
cartridges (NEN-DuPont) prior to use. Oligonucleotides used are listed in
Table 1.
TABLE 1
__________________________________________________________________________
Oligonucleotides Used for
PCR Amplifications and Sequencing
No. Sequence 5' Coordinate
Ref.
__________________________________________________________________________
228A
AACAGCTATGACCATGAT pUC polylinker
SEQ ID NO:1
229
TTCCCAGTCACGACGTTG pUC polylinker
SEQ ID NO:2
230
TGTAAAACGACGGCCAGT pUC polylinker
SEQ ID NO:3
1138
AGGGATTGGTTGGCCTGGTCCTCCGAA
637(+), 16S
3 SEQ ID NO:4
1887
CTTTCCAGTAATAGGCTTATCG
1726(-), 16S
3 SEQ ID NO:5
2892
GCTATCCTGAGGGAAACTTCGG
964(-), 26S SEQ ID NO:6
2893
CCCGTCTTGAAACACGGACCAAGG
635(+), 26S SEQ ID NO:7
2894
CCCGCGATCAGCAAAAGCTAATCTGG
1374(-), 16S
3 SEQ ID NO:8
2917
CCATACAGAAGACCATTCTTTATCCC
507(-), DHFR
18 SEQ ID NO:9
2918
GGCCGATCAAACTCTCTTCC
58(+), DHFR
18 SEQ ID NO:10
2919
GGGAAAAGGTCGTGGGGAGCG
977(-), TS
17 SEQ ID NO:11
2920
GGGGAAGACCGCCCTGATAGG
58(+), TS
17 SEQ ID NO:12
2982
GAGCCAATCCTTATCCCGAAGTTACG
1933(-), 26S
SEQ ID NO:13
2983
GTCTAAACCCAGCTCACGTTCCC
2933(-), 26S
SEQ ID NO:14
3175
GGGTGGTGGTGCATGGCCG
1262(+), 16S
3 SEQ ID NO:15
3176
CCTTCCGCAGGTTCACCTACGG
1796(-), 16S
3 SEQ ID NO:16
3243
CCGCAGCAGGTCTCCAAG 1833(+), 26S
SEQ ID NO:17
3425
CGAAAGAGAGGAGGTAGCACC
368(+), intron, 16S
5 SEQ ID NO:18
3426
GGTCCGTGTTTCAAGACGGG
654(-), 26S SEQ ID NO:19
3427
GGGAACGTGAGCTGGGTTTAG
2911(+), 26S
SEQ ID NO:20
4016
GGTTTGGCAGGCCAACATCGG
485(+), 26S SEQ ID NO:21
4138
CCATGAAAGTGTGGCCTATCG
2715(+), 26S
SEQ ID NO:22
4139
GCCTGGTCAGACAACCGC 3049(-), 26S
SEQ ID NO:23
4169
GGATTATGGCTGAACGCC 3074(+), 26S
SEQ ID NO:24
4170
GGCTTAATCTCAGCAGATCG
3328(-), 26S
SEQ ID NO:25
4358
GACGAGGCATTTGGCTACC
2267(-), 26S
SEQ ID NO:26
4443
GTACACACCGCCCGTCGC 1631(+), 16S
3 SEQ ID NO:27
4743
TTTAGCTCTTGATTGTAG 556(+), 26S, Pc2
SEQ ID NO:28
4744
CGCATATTTTATATTATG 3234(-), 26S, Pc2
SEQ ID NO:29
4746
GTTAGCTCTTGGCTTCTG 556(+), 26S, Pc1
SEQ ID NO:30
__________________________________________________________________________
TS refers to the thymidylate synthase (17) and DHFR refers to the
dihydrofolate reductase (18) genes of P. carinii.
Table 1 lists all primers used for PCR amplifications and sequencing. The
underlined G in 3243 was predicted for the 26S rRNA gene sequence based on
sequences from other organisms, but was A in the actual 26S rRNA sequence
of P. carinii. The underlined C in 4169 was present in the 26S rRNA gene
of P. carinii from Hooded rats but was A in the homologous location in
organisms from Sprague-Dawley rats, as described in the text. The
underlined C in 3425 is from the published intron sequence (5) but was T
in a clone of the intron amplified using flanking exon-derived primers
4434 and 3176.
Table II shows the extent of genetic identity as indicated by the
Wisconsin-GCG "Distances" program. Sequences are from GenBank with the
following accession numbers: Neurosopora crassa, Nc X02447; Cephalosporium
acremonium, Ca X06574; Alternaria alternata, Aa X17454; Saccharomyces
cerevisiae, Sc K01051; Schizosaccharomyces pombe, Sp J01359; Pneumocystis
carinii, Pc; Acanthamoeba castellani, Ac K00471; Chlamydomonas
reinhardtii, Cr M35013; Tetrahymena pyriformis, Tp M10752; Trypanosoma
brucei, Tb X05682; Plasmodium falciparum, Pf J04683; Dictyostelium
discoideum, Dd V00192; Phyarum polycephalum, Pp M13612; and Giardia
lamblia, Gl M35013.
TABLE II
__________________________________________________________________________
Sequence Similarity of 5.8S rRNAs of Simple Eukaryotes
Nc Ca Aa Sc Sp Pc Ac Cr Tp Tb Pf Dd Pp Gl
__________________________________________________________________________
Nc
1.0000
.9299
.9236
.9172
.8599
.8854
.7771
.7308
.6883
.6624
.5159
.5414
.5097
.4483
Ca 1.0000
.8924
.8797
.8544
.8418
.7215
.7244
.6688
.6519
.4873
.5506
.4968
.4828
Aa 1.0000
.9494
.8987
.8671
.7722
.7436
.6883
.6582
.5380
.5506
.5161
.4483
Sc 1.0000
.9114
.8734
.7848
.7564
.7143
.6392
.5316
.5696
.5161
.4483
Sp 1.0000
.8165
.7407
.7500
.7143
.5879
.5273
.5432
.5290
.4759
Pc 1.0000
.7468
.7051
.6753
.6519
5063
.5443
.5032
.4207
Ac 1.000
.7500
.6818
.5679
.5185
.5000
.5032
.4828
Cr 1.0000
.6429
.5641
.5513
.4744
.4516
.4552
Tp 1.0000
.5844
.5714
.5130
.5000
.4414
Tb 1.0000
.4702
.4691
.5161
.4138
Pf 1.0000
.4753
.4452
.3793
Dd 1.0000
.4065
.3862
Pp 1.0000
.4483
Gl 1.0000
__________________________________________________________________________
TABLE III
______________________________________
Sequence Similarity of 26S rRNAs of Simple Eukaryotes
Pc Sc Tp Pp
______________________________________
Pc -- 0.833 0.739
0.623
Sc -- 0.734
0.602
Tp -- 0.605
______________________________________
Table III shows the extent of genetic identity of 26S rRNA gene sequences,
calculated as in Table II. Abbreviations are as in Table II; sequences
from GenBank include Sc, J01355; Tp, X54004; and Pp, V01159.
Amplification and Cloning of DNA
Pneumocystis carinii DNA was amplified by means of PCR performed in a DNA
Thermal Cycler (Perkin Elmer Cetus) using thermostable DNA polymerase from
Thermus aquaticus (AmpliTaq, Perkin Elmer Cetus). Reactions were run in
the presence of 0.2 mM of each dNTP, 0.4 uM of each of the indicated
primers, 10 mM Tris-HCl (pH 8.3), 50 mM potassium chloride, 1.5 mM
MgCl.sub.2, gelatin (0.001% w/v), and 5 units of AmpliTaq DNA polymerase
in 10 ul total volume. Amplifications of segments over 1 kb. were
performed by incubation at 95.degree. C. for 2 minutes followed by 30
cycles of 94.degree. C. for 1 minute, 50.degree. C. for 1 minute, and
72.degree. C. for 1.5 minutes, followed by a 7 minute incubation at
72.degree. C. Amplifications of fragments of less than 1 kb. were
performed by 2 cycles of 94.degree. C. for 2 minutes, 58.degree. C. for 1
minute, and 72.degree. C. for 45 seconds, followed by 30 cycles of
94.degree. C. for 1 minute, 58.degree. C. for 1 minute, and 72.degree. C.
for 1 minute, followed by incubation at 72.degree. C. for 1 minute. For
some PCR reactions, the thermostable DNA polymerase from Thermus
thermophilus (Hot Tub, Amersham) was used, under reaction conditions
recommended by the manufacturer using 1.5 units of polymerase in a 100 ul
reaction, using 2 cycles of 94.degree. C. for 2 minutes, 58.degree. C. for
1 minute, and 70.degree. C. for 2 minutes, followed by 30 cycles of
94.degree. C. for 1 minute, 59.degree. C. for 1 minute, and 70.degree. C.
for 3 minutes, followed by incubation at 70.degree. C. for 10 minutes.
After PCR reaction, products were purified by agarose gel electrophoresis,
treated with T4 DNA polymerase (BRL) to generate blunt ends,
phosphorylated with T4 polynucleotide kinase (Pharmacia), ligated under
blunt end ligation conditions to SmaI-cut pUC18 DNA, and transformed into
E. coli DH5-alpha competent cells (BRL, Bethesda, Md.) as described (19).
Cells were grown in LB medium and plasmid DNA was extracted and purified
as described (19).
DNA Sequence Determination
DNA sequence determination was performed on the Genesis 2,000 Automated DNA
Sequencer (DuPont) according to the manufacturer's instructions for
sequencing reactions run on covalently closed superhelical DNA templates,
using DNA polymerase from bacteriophage T7 (Sequenase version 1.0, U.S.
Biochemicals). Primers used included oligonucleotides 228A, 229, and 230
(Table 1), which base pair with regions flanking the pUC18 polylinker, and
others listed in Table 1. For inserts of over 300 nucleotides without
convenient internal primer binding sites, nested deletions were generated
as described (19), which were then sequenced using the standard primers.
All sequences reported were determined at least twice for each DNA strand.
RESULTS
Sequence of the rRNA Operon of P. carinii
Prior to use for these experiments, nucleic acids from P. carinii were
shown to be from that source by confirmation of previously published
sequences using PCR methods. Primers 2920 and 2919 used in a PCR reaction
yielded a single 920 bP. product (based on agarose gel electrophoresis),
the size predicted for the thymidylate synthase gene with its 4
intervening sequences (17). A PCR utilizing primers 2918 and 2917
amplified a single 493 bp. product, as predicted for the dihydrofolate
reductase gene with a 43 bp. intervening sequence (18). The P.
carinii-specific primers for 16S rRNA, 1138 and 2894, yielded a single PCR
product of the predicted 738 bp. size (3). The "universal" 16S rRNA
primers, 3175 and 3176, generated two PCR products: one was 925 bp. in
length, the size predicted for the 16S rRNA gene with its Group I intron
(3, 5), and the other was 535 bp. in length. This smaller fragment had a
sequence identical to the corresponding region of human 18S rRNA (21), and
presumably represents amplification of contaminating mink lung cell
ribosomal DNA. The sequence of mink 16S rRNA is unknown, but is presumably
closely related to the human sequence.
FIG. 1 shows the DNA sequence of a portion of the rRNA-encoding gene(s) of
P. carinii isolated from immunosuppressed Sprague-Dawley rats (Sasco) and
the PCR amplifications which were subsequently cloned and sequenced. The
top line represents the DNA sequence of a portion of the rRNA-encoding
gene(s) of P. carinii isolated from immunosuppressed Sprague-Dawley rats
(Sasco). The horizontal lines below represent PCR amplifications which
were subsequently cloned and sequenced. Thin lines (FIG. 1A) refer to PCR
products from Sprague-Dawley rats (Sasco) and heavy lines (FIG. 1B) refer
to PCR products from Hooded rats. Numbers refer to oligonucleotide primers
(Table 1) used in each PCR reaction. Each PCR product, produced using
primers listed in Table 1, was cloned into pUC18 and both strands were
sequenced at least twice. All overlapping segments yielded the same
sequence, indicating an error rate of Taq polymerase-catalyzed PCR (22) of
less than one per 500 nucleotides. Rare misincorporation events in the
regions which were only amplified once cannot be ruled out.
FIG. 2 shows the total contiguous sequence determined for P. carinii from
immunosuppressed Sprague-Dawley rats (Sasco) by the strategy shown in FIG.
1A. Except for the last 18 nucleotides (shown in lower case), capital
letters indicate rRNA coding sequences (positive strand), lower case
letters indicate spacers, and underlined lower case letters indicate Group
I introns. The initial 22 nucleotides are from the 3'-terminal portion of
the Group I intron in 16S rRNA. Nucleotides 23-53 are the second exon of
16S rRNA, 54-216 are internal transcribed spacer 1 (ITS1), 217-374 the
gene for 5.8S rRNA (identified by similarity to other 5.8S rRNA
sequences), 375-556 ITS2, and 557-4256 are the gene for 26S rRNA, with a
Group I intron sequence in lower case underlined. This sequence has been
deposited at EMBL/GenBank under accession No. M86760. The sequence of the
final exon of the 16S rRNA gene agrees with that previously reported (3),
although the third base from the 3' end of the intron (C) previously
reported (5) is absent in our sequence. This sequence has been confirmed
in an additional amplified fragment including the entire intron sequence.
FIG. 3 shows a comparison of the sequence of the 5.8S rRNA gene of P.
carinii shown in FIG. 2 with the homologous sequences from Saccharomyces
cerevisiae (23) shown as Sc, Tetrahymena pyriformis (24) shown as Tp, and
Homo sapiens (25) shown as Hs. Since the actual 5.8S rRNA sequence was not
determined, the termini of the P. carinii gene have been chosen based on
the known sequence of the homologous gene of S. cerevisiae, to which it
appears to be closely related. The three nucleotides 5' to the proposed
rRNA 5' terminus are shown here in lower case letters. The 5.8S rRNA
sequence is 87% identical with the homologous rRNA of S. cerevisiae, which
was also the species to which P. carinii showed closest relatedness of its
16S rRNA gene (3). In contrast, the 5.8S rRNA sequence was 67% and 69%
identical with the homologous genes of T. pyriformis and H. sapiens,
respectively.
FIG. 5 shows the sequence of the 26S rRNA gene from FIG. 2 compared to
homologous genes from S. cerevisiae (26) and T. pyriformis (27). The
indicated P. carinii sequence has an apparent Group I self-splicing intron
sequence (see below) omitted after nucleotide 2241, and the T. pyriformis
sequence has an intron of the same type omitted from a location four
nucleotides 3' to the homologous site in the P. carinii gene (27). The
final 18 nucleotides of the P. carinii sequence were determined from
organisms from immunosuppressed Hooded rats as shown in FIG. 2. Thus the
26S rRNA genes of both P. carinii and T. pyriformis have Group I
self-splicing introns inserted into the same relatively conserved region.
Comparison of the three sequences shown in FIG. 5 indicates the relative
conservation of some regions of the 26S rRNA genes, and the greater
phylogenetic variability of other regions. The sequence of the coding
region of the P. carinii 26S rRNA gene shown in FIG. 5 is 83.3% identical
with the homologous gene of S. cerevisiae and 73.9% identical with that of
T. pyriformis. Therefore, based upon all three genes (encoding 16S, 5.8S
and 26S rRNA) of the major rRNA operon, P. carinii appears to be more
closely related to S. cerevisiae than to representative "protozoa."
Group I Self-splicing Introns of rRNA Genes
As set out in FIG. 2, an apparent Group I self-splicing intron interrupts
the 26S rRNA gene sequence in P. carinii. This intron is recognizable by
the presence of the conserved P, Q, R, and S segments (boldface in FIG.
6A)) present in all introns of this class, as previously reviewed (6-7).
There is 74% identity between the sequence of the putative Group I intron
in the 26S rRNA gene and that previously reported (5) in the 16S rRNA
gene. The entire sequence of the 16S rRNA gene intron in the P. carinii
isolate has been confirmed, and is identical to that reported (5) except
for the absence of the third nucleotide from the 3' end of the intron (C).
FIG. 6A shows the secondary structure into which the apparent Group I
intron in the gene for 26S rRNA of P. carinii can be folded. The helices
P1-P9 are conserved among Group I introns (6-7). The bases in the intron
are numbered 1 through 355, and the flanking exon regions are shown in
lower case letters. The consensus sequences P (nucleotides 80-91), Q
(nucleotides 202-211), R (nucleotides 247-260) and S (nucleotides 316-327)
are shown in boldface. FIG. 6B shows an alternative folding for the P8
helix of the intron (5) in the 16S rRNA gene.
FIG. 6A shows that the 26S rRNA gene intron can be folded into a structure
similar to that reported for other Group I self-splicing introns (6-7),
including that in the gene encoding 16S rRNA in P. carinii (5). This
structure is not necessarily the most stable folded structure possible
(28), but is most consistent with the consensus folding proposed for Group
I introns (7). The structure in FIG. 6A contains the conserved P1
double-helix made up of a pairing of the 5' exon-intron junction with an
internal guiding intron sequence (IGS). It also contains an unusually long
P8 helix with a bulge-loop on its 5' side. Although the previously
proposed structure for the 16S intron (5) does not have such an elongated
P8 helix, its structure also can be drawn in this way (FIG. 6B).
PCR primers pairing to the exons on either side of the 26S rRNA gene intron
were utilized, including a 5' primer with a 17-nucleotide 5' extension
consisting of a bacteriophage SP6 promoter (29), to generate a DNA product
consisting of the intron sequence with portions of both flanking exons
with an SP6 promoter at the 5' end of the positive strand. Transcription
of this DNA by bacteriophage SP6 RNA polymerase (Promega) results in
production of RNA catalyzing self-splicing under similar conditions to
those reported (5) for self-splicing of the intron in the 16S rRNA gene.
Thus the three rRNA genes encoding 16S, 5.8S and 26S rRNA of P. carinii
closely resemble their homologues in S. cerevisiae in sequence. However,
they contain Group I self-splicing introns in the 16S and 26S rRNA genes,
unlike most known fungi but like some protozoa (27).
Sequence Variation between P. carinii Isolates
In the course of studies to confirm the sequence shown in FIG. 2, various
regions of the rRNA operon of P. carinii were repeatedly amplified and
sequenced. Organisms obtained from the lungs of Sprague-Dawley rats
(Sasco) immunosuppressed in isolation chambers yielded the same sequences
for duplicate or overlapping amplifications, as summarized in FIG. 1. When
portions of the 26S rDNA were amplified, cloned and sequenced from P.
carinii obtained from Hooded rats immunosuppressed without isolation, they
were found to differ in sequence from the same regions obtained from
organisms from Sprague-Dawley rats from Sasco (FIGS. 7 and 8).
FIG. 7 shows the sequence of a region of the 26S rRNA gene which was
determined for five independent PCR products (summarized in FIG. 1) using
three different sets of primers from P. carinii from Sprague-Dawley rats,
for the region of nucleotides 485-964 as shown in FIG. 5. This sequence is
denoted Pc1 in FIG. 7, and was identical in all five determinations,
including three derived using PCR primers shown by the underlined
sequences in FIG. 7 and two using one primer outside this region and one
within it, as shown in the legend of FIG. 7. When the pair of primers
shown in FIG. 7 was used to amplify DNA from P. carinii from Hooded rats,
the sequence shown as Pc2 was obtained. Comparison of these sequences with
those of S. cerevisiae and T. pyriformis 26S rRNA sequences demonstrates
that the DNA sequences of the two P. carinii isolates differ from each
other at multiple positions, with the differences occurring mostly in
phylogenetically variable regions of the rRNA sequence. However, the two
P. carinii sequences are clearly more similar to each other than to the
sequence of the S. cerevisiae gene, indicating the phylogenetic
relatedness of these two isolates.
FIG. 8 shows a comparison of the sequences of the region from nucleotides
2911 through 3327 of the 26S rRNA gene of P. carinii (Pc1) from
Sprague-Dawley rats (FIG. 5) with the homologous regions from P. carinii
from Hooded rats (Pc2) and from S. cerevisiae (Sc) and T. pyriformis (Tp).
The fragment denoted Pc1 was amplified using primers 4138 and 4170. The
sequence shown for Pc2 was determined based on amplifications using primer
pair 4138 and 4139 and pair 4169 and 4170, and ligation-dependent PCR
amplification of a fragment extending from oligonucleotide 3427 through a
PstI site 381 nucleotides past the 3' end of the 26S rRNA gene. The
sequences of homologous regions of the 26S rRNA genes of S. cerevisiae
(Sc) and T. pyriformis (Tp) are shown. The 3'-terminal region of the 26S
rRNA gene of P. carinii from these two sources differed from each other,
with most of the differences in phylogenetically non-conserved regions.
Again the two P. carinii genes showed greater similarity to each other
than to the genes from other species.
When Pc1 DNA template was amplified by PCR using the primer pair 4358
(universal) and 4746 (Pc1-specific), the expected 2,067 bp product was
produced. In contrast, no product was generated from Pc2 template with
these same primers (FIG. 9). Similarly, primers 4743 (Pc2-specific) and
4744 (Pc2- specific) amplified an approximately 3.0 kbp product from Pc2
template; no similar product was seen with Pc1 template (FIG. 9). Note
that in some reactions a barely detectable band of the same size seen with
Pc2 template was seen with Pc1 template using the latter primer pair.
These data are consistent with Pc1 and Pc2 each containing predominantly
genes encoding single distinct major 26S rRNA sequences.
External Transcribed Spacer Sequence
The sequence of the 26S rRNA gene shown in FIG. 3 contains a
phylogenetically conserved EcoRI site at position 2875, which is located
in a highly conserved region of the sequence. DNA isolated from P. carinii
from Hooded rats was restricted with pairs of restriction enzymes,
including EcoRI and various other "6-cutters," and the resulting fragments
were then ligated into pUC18 cut with the same pairs of restriction
enzymes. The product of each of the ligation reactions was then subjected
to PCR amplification, with thermostable DNA polymerase from Thermus
thermophilus (Hot Tub, Amersham) using the primer pair: oligonucleotide
3427, which pairs on the positive strand at positions 2911-2931, and
oligonucleotide 230, which pairs with a pUC18 region 3' to the polylinker
(on the negative strand). When such PCR reactions were analyzed by agarose
gel electrophoresis with visualization of bands by ultraviolet
light-induced fluorescence in the presence of ethidium bromide, only the
pair of restriction enzymes EcoRI and PstI generated a visible DNA band.
When this band was cloned and sequenced, its 5' region had the sequence
shown as Pc2 in FIG. 8, followed by the final 18 nucleotides of the 26S
rRNA gene as shown in FIG. 5 and 381 nucleotides of the following spacer
region shown in FIG. 10, which would correspond to the external
transcribed spacer region in the homologous operon of most eukaryotes
(reviewed in 30). When the same ligation-dependent PCR procedure was
followed using the DNA from P. carinii from Sprague-Dawley rats, no
visible band of DNA was detected. This presumably indicates that the PstI
site in the spacer of the DNA denoted Pc2 is absent in Pc1 DNA, and the
next one is presumably too distant to support ligation-dependent PCR.
FIG. 10 shows the sequence of the spacer region 3' to the 26S rRNA gene of
P. carinii from Hooded rats (FIG. 8), which was determined by
ligation-dependent PCR. The sequences shown in FIGS. 8 and 10 have been
deposited at EMBL/GenBank under accession No. 86759.
In accord with the present invention, a method is provided for diagnosing
for Pneumocystis carinii which comprises detecting the presence of a
nucleic acid sequence containing the 26S rRNA gene specific for
Pneumocystis carinii in a sample which comprises the steps of:
(a) treating the sample with an oligodeoxyribonucleotide primer for each
strand of the nucleic acid sequence, four different nucleoside
triphosphates, and an agent for polymerization under hybridizing
conditions, such that for each strand an extension product of each primer
is synthesized which is sufficiently complementary to each strand of the
nucleic acid sequence being detected to hybridize therewith and contains
the 26S rRNA gene specific for Pneumocystis carinii, wherein the primers
are selected such that the extension product synthesized from one primer,
when it is separated from its complement, can serve as a template for
synthesis of the extension product of the other primer;
(b) treating the sample from step (a) under denaturing conditions to
separate the primer extension products from the templates on which they
are synthesized if the sequence to be detected is present;
(c) treating the product from step (b) with oligodeoxyribonucleotide
primers, four different nucleoside triphosphates, and an agent for
polymerization such that a primer extension product is synthesized using
each of the single strands produced in step (b) as a template, resulting
in amplification of the sequence to be detected if present;
(d) hybridizing the primer extension products from step (c) with a labeled
oligodeoxyribonucleotide probe complementary to the 26S rRNA gene specific
for Pneumocystis carinii;
(e) determining whether hybridization in step (d) has occurred.
Amplified products may be detected by electrophoresis on agarose gels
followed by hybridization with a radioactive or nonradioactive probe
consisting of a third oligonucleotide specific for a sequence lying
between two PCR primers on the P. carinii gene. The method may further
comprise in steps (d) and (e) a positive control which contains the 26S
rRNA gene specific for Pneumocystis carinii and a negative control which
does not contain the 26S rRNA gene.
This invention also provides a method for diagnosing for various species of
P. carinii by detecting the presence of a nucleic acid sequence containing
the particular 16S or 26S rRNA gene sequence specific for that species of
P. carinii. Specific PCR primers and hybridization probes for specific
subtypes of P. carinii may be employed based on sequence analysis of
different subtypes found in infected rats. Alternatively, single pairs of
PCR primers based on sequences shared by all isolates may be used for
strain identification if the distances between sequences shared by
different isolates are distinct. This latter approach may prove useful if
different strains differ in the location of the introns in their genes.
Preliminary data show that this is the case for the introns in 16S rRNA in
the two rat-derived P. carinii isolates described above.
Methods for amplifying and detecting nucleic acid sequences are described
in detail in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, which
disclosures are incorporated herein by reference.
The present invention is also directed at methods for diagnosing for
Pneumocystis carinii which comprise detecting the presence of RNA
complementary to a nucleic acid sequence containing the 26S rRNA gene
specific for Pneumocystis carinii, the 26S rRNA gene specific for a
species of Pneumocystis carinii, and the 16S rRNA gene specific for a
species of Pneumocystis carinii. The methods involve using PCR to amplify
mRNA sequences from cDNA. In this method, the enzyme reverse transcriptase
and a primer specific for the RNA are employed to make a DNA copy of the
RNA. The DNA copy may then be amplified and detected by the methods of the
present invention. Examples of reverse transcriptase enzymes which may be
employed include Moloney murine leukemia virus (MuLV) and Avian
Myeloblastosis virus (AMV) enzymes. Methods for employing PCR to amplify
mRNA sequences from cDNA are more fully described in G. Veres et al.,
Science, 237:415-417 (1987) and PCR Protocols; A Guide to Methods and
Applications, Edited by M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T.
J. White, Academic Press, 1990, pp. 21-27, which disclosures are
incorporated herein by reference.
Appendium of References
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Pediatrics, 61, 35-41.
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Cushion, M. T. (1989) Exptal. Parasitol., 68, 450-461.
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Biochem. Parasitol., 32, 163-168.
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(1989) Infect. Immun., 57, 2149-2157.
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(1988) J. Clin. Microbiol., 26, 774-775.
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Natl. Acad. Sci. USA, 86, 6503-6507.
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Santi, D. V. (1989) Proc. Natl. Acad. Sci. USA, 86, 8625-8629.
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Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor
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20. Torczynski, R. M., Fuke, M., and Bollon, A. P. (1985) DNA, 4, 282-291.
21. Jones, M. D., and Foulkes, N. S. (1989) Nucleic Acids Res., 17,
8387-8388.
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23. Bell, G. I., Degennaro, L. J., Gelfand, D. H., Bishop, R. J.,
Valenzuela, P., and Rutter, W. J. (1977) J. Biol. Chem., 252, 8118-8125.
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505-508.
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Acids Res., 9, 6953-6958.
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28. Zuker, M., and Stiegler, P. (1981) Nucleic Acids Res., 9, 133-148.
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30. Musters, W., Planta, R. J., van Heerikhuizen, H., and Raue (1990) in
Hill, W. E., Dahlberg, A., Garrett, R. A., Moore, P. B., Schlessinger, D.,
and Warner, J. R. (eds.), The Ribosome, Amer. Soc. Microbiol., New York,
pp. 435-442.
31. van Ahsen, U., Davies, J., and Schroeder, R. (1991) Nature, 353,
368-370.
32. Vossbrinck, C. R., Maddox, J. V., Friedman, S., Debrunner-Vossbrinck,
P. A., and Woese, C. R. (1987) Nature, 326, 411-414.
33. Kim, H. K., Hughes, W. T., and Feldman, S. (1972) Proc. Soc. Exptal.
Biol. Med., 142, 304-309.
34. Walzer, P. D., and Rutledge, M. E. (1980) J. Infect. Dis., 142, 449.
35. Gigliotti, F., Stokes, D. C., Cheatham, A. B., Davis, D. S., and
Hughes, W. T. (1986) J. Infect. Dis., 154, 315-322.
36. Link, M. J., Cushion, M. T., and Walzer, P. D. (1989) Infect. Immun.,
57, 1547-1555.
37. Tanabe, K., Fuchimoto, M., Egawa, K., and Nakamura, Y. (1988) J.
Infect. Dis., 157, 593-596.
38. Hughes, W. T., and Gigliotti, F. (1988) J. Infect. Dis., 157, 432-433.
39. Gunderson, J. J., Sogin, M. L., Wollett, G., Hollingdale, M., de la
Cruz, V. F., Waters, A. P., and McCutchan, T. F. (1987) Science, 238,
933-937.
40. Gonzalez, I. L., Gorski, J. L., Campen, T. J., Dorney, D. J., Erickson,
J. M., Sylvester, J. E., and Schmickel, R. D. (1985) Proc. Natl. Acad.
Sci. USA, 82, 7666-7670.
41. van Keulen, H., Campbell, S. L., Erlandsen, S. L., and Jarroll, E. L.
(1991) Mol. Biochem. Parasitol., 46, 275-284.
Throughout this application, various publications have been referenced. The
disclosures in these publications are incorporated herein by reference in
order to more fully describe the state of the art.
The invention being thus described, it will be obvious that the same may be
varied in many ways. Such variations are not to be regarded as a departure
from the spirit and scope of the invention and all such modifications are
intended to be included within the scope of the following claims.
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 32
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
AACAGCTATGACCATGAT18
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
TTCCCAGTCACGACGTTG18
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
TGTAAAACGACGGCCAGT18
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
AGGGATTGGTTGGCCTGGTCCTCCGAA27
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CTTTCCAGTAATAGGCTTATCG22
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GCTATCCTGAGGGAAACTTCGG22
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CCCGTCTTGAAACACGGACCAAGG24
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CCCGCGATCAGCAAAAGCTAATCTGG26
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
CCATACAGAAGACCATTCTTTATCCC26
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GGCCGATCAAACTCTCTTCC20
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
GGGAAAAGGTCGTGGGGAGCG21
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
GGGGAAGACCGCCCTGATAGG21
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GAGCCAATCCTTATCCCGAAGTTACG26
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
GTCTAAACCCAGCTCACGTTCCC23
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
GGGTGGTGGTGCATGGCCG19
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
CCTTCCGCAGGTTCACCTACGG22
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
CCGCAGCAGGTCTCCAAG18
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
CGAAAGAGAGGAGGTAGCACC21
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
GGTCCGTGTTTCAAGACGGG20
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
GGGAACGTGAGCTGGGTTTAG21
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
GGTTTGGCAGGCCAACATCGG21
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
CCATGAAAGTGTGGCCTATCG21
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
GCCTGGTCAGACAACCGC18
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
GGATTATGGCTGAACGCC18
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
GGCTTAATCTCAGCAGATCG20
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
GACGAGGCATTTGGCTACC19
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
GTACACACCGCCCGTCGC18
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
TTTAGCTCTTGATTGTAG18
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
CGCATATTTTATATTATG18
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
GTTAGCTCTTGGCTTCTG18
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4256 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
CGAAAGAGAGGAGGTAGCACCGTTCCGTAGGTGAACCTGCGGAAGGATCATTAATGAAAT60
GTTGTCAAGAACTAGTTTATCTGGTTCTTGACATTTTCATCATAACACTTGTGAACATTA120
AAGATTTGCTTTGACAGGATGGGAGTTAGCTTTCGTCCTGTCAGAGGTTTTCAATTAAAA180
CTTTTTTGGTGTTTCGGTTAAAAATATAATTTTTAAAAACTTTCAGCAATGGATCTCTTG240
GTTCCCGCGTCGATGAAGAACGTGGCAAAATGCGATAAGTAGTGTGAATTGCAGAATTCA300
GTGACTCATCGAATTTTTGAACGCATATTGCGCTCCTCAGTATTCTGTGGAGCATGCCTG360
TTTGAGCGTCATTTTTATACTTGAACCTTTTTAAGGTTTGTGTTGGGCTATGCATTTTAG420
TATTTTTACAAGATGCTAGTCTAAAATGGAATCCAGAATATTATTTCGTGCAGCGTAATA480
GGGTTAAATTCCAATTCGCTGTTTTTAGAAATGATAGACTGGTTTGTCTATTGTTCCTAG540
AGAGCAATTTTTGAACCTTTGACCTCAAATCAGGTAGGATTACCCGCTGAACTTAAGCAT600
ATCAATAAGCGGAGGAAAAGAAACTAACAAGGATTCCCTCAGTAACGGCGAGTGAAGTGG660
GAAAAGCTCAAAATTAAAATCTGGCGAGGATCCTCGTCCGAGTTGTAATTTAGAGAAGTG720
CTTTTGGCTTGATGCTCTATTTAAAGTCCTTTGGAACAAGGCATCATAGAGGGTGATAAT780
CCCGTACGAGTAGGGTTATTAAGCTATGTAAAAGCACATTCGAAGAGTCGAGTTGTTTGG840
GATTGCAGCTCAAAATGGGTGGTAAATTTCATCTAAAGCTAAATATTAGCGGGAGACCGA900
TAGCGAACAAGTAGAGTGATCGAAAGATGAAAAGAACTTTGAAAAGAGAGTTAAATAGTA960
CGTGAAATTGCTGAAAGGGAAGCGCTTGCGATCAGACATGCCTTATCAGGATGTTGTTGT1020
CTTGACAATAACTATTACTTGGTTTGGCAGGCCAACATCGGTTTCAGCTGCTAGGTAAGT1080
GTCAAGAGAGGGTAGCCTCTTTCGTGGGGTGGTTAGCTCTTGGCTTCTGTAGTAGCAGGG1140
ACCGGAAGGTCTAGCGTCAGCTTGGTTGTTGGCTTAATGGTCTTAAGCGACCCGTCTTGA1200
AACACGGACCAAGGAGTCTAATATCTATGCGAGTGTTTGAGTGGAAAACTCATACGCGAA1260
ATGAAAGTGAAGCAAAAGGTAGGAACCCTTTAAGGGTGCACTATCGACCGGTTCAAATTT1320
ATTTGGATTGAGTAAGAGCATAGCTATTGGGACCCGAAAGATGGTGAACTATGCCTGAAT1380
AGGGTGAAGCCAGAGGAAACTCTGGTGGAGGCTCGTAGCGGTTCTGACGTGCAAATCGAT1440
CGTCAAATTTGGGCATAGGGGCGAAAGACTAATCGAACCATCTAGTAGCTGGTTCCTGCC1500
GAAGTTTCCCTCAGGATAGCAGAAACTCAATATCAGTTTTATGAGGTAAAGCGAATGATT1560
AGAGGCATTGGGGTTGAAACAACCTTAACCTATTCTCAAACTTTAAATATGTAAGAAGTC1620
CTTGTTGCTTAATTGAACATGGACATTAGAATGAGAGTTTCTAGTGGGCCATTTTTGGTA1680
AGCAGAACTGGCGATGCGGGATGAACCGAACGCGAGGTTAAGGTGCCGGAAGCACGCTCA1740
TCAGATACCACAAAAGGTGTTAGTTCATCTAGACAGTAGGACGGTGGCCATGGAAGTCGG1800
AATCCGCTAAGGAGTGTGTAACAACTCACCTACCGAATGAACTGGCCCTGAAAATGGATG1860
GCGCTCAAGCGTGCTACCTATACCTCGCCGTCTGGGATAATGATTCCTAGACGAGTAGGC1920
AGGCGTGGGGGTCGTGGCGAAGCCTAGGGCGTGAGCCCGGGTTGAACGGCCTCTAGTGCA1980
GATCTTGGTGGTAGTAGCAAATATTCAAATGAGGACTTTGAAGACTGAAGTGGGGAAAGG2040
TTCCATGCGAACAGTTATTGGGCATGGGTTAGTCGATCCTAAGAGATAGGGAAACTCCGT2100
TTTAAAGTGCGCGATTTTTCGCGCCTCTATCGAAAGGGAATCCGGTTAATATTCCGGAAC2160
CAGGATATGGATTCTTCACGGCAACGTAAATGAAGTCGGAGACGTCAGCGGGGGGCCTGG2220
GAAGAGTTATCTTTTCTTCTTAACAGCCTATCACCCTGGAATCGGTTTATCCGGAGATAG2280
GGTTCAATGGCTGGTAGAGTTCAGCACTTCTGTTGAATCCAGTGCGCTTTCGATGACCCT2340
TGAAAATCCGACGGAAGGAATAGTTTTCATGCCTGGTCGTACTCATAACCGCAACAGGTC2400
TCCAAGGTGAACAGCCTCTAGTTGATAGAATAATGTAGATAAGGGAAGTCGGCAAAATAG2460
ATCCGTAACTTCGGGATAAGGATTGGCTCTAAGGATTGGGTGCATTGGGCTTTAATCGGA2520
AGCTATTGGACCAGACGGGAACTACCTTGGGAAACCGAGGCGGATCCTGTTAGGATCGAT2580
CAGTGAATGATTTTAGCAGCCCTTTGGGCGTCCGATGCACGCTTAACAATCAACTTAGAA2640
CTGGTACGGACAAGGGGAATCTGACTGTCTAATTAAAACATAGCATTGCGATGGCCAGAA2700
AGTGGTGTTGACGCGATGTGATTTCTGCCCAGTGCTCTGAATGTCAAAGTGAAGAAATTC2760
AACCAAGCGCGGGTAAACGGCGGGAGTAACTATGACTCACCTTTTGAGGGTCATGAAAGC2820
GGCGCGAAAGTGTTAGCTAGTGATCCGAAAAATAAATTCGGGTTGCGACACTGTCAAATT2880
GCGGGGAGTCCCTAAAGATTCAACTACTAAGCAGCTTGTGGAAACACAGTTGTGGCCGAG2940
TTAATAGCCCTGGGTATAGTAACAATGTTGAATATGACTCTTAATTGAGGAAATGGGTGA3000
TCCGCAGCCAAATCCTAAGGACATTTTATTGTCTATGGATGCAGTTCAGCGACTAGACGG3060
CAGTGGGTATTGTAGAGATATGGGGTTATTTATGGCCTTATCTACAATGCTTAAGGTATA3120
GTCTAATCTCTTTCGAAAGAAAGAGTAGTGTGCTCTTAAGGTAGCCAAATGCCTCGTCAT3180
CTGATTAGTGACGCGCATGAATGGATTAACGAGATTCCCACTGTCCCTATCTACGATCTA3240
GCGAAACCACAGCCAAGGGAATGGGCTTGGCAAAATCAGCGGGGAAAGAAGACCCTGTTG3300
AGCTTGACTCTAGTTTGACATTGTGAAAAGACATAGAGGATGTAGAATAGGTGGGAGCTT3360
CGGCGCCTGTGAAATACCACCGCCTTTATTGTTTTTTTACTTAATCAGTGGAGCGGGACT3420
GAGCTTTTGCTCATCTTTTAGCGTTAAGGTCCTTTTACGGGCCGACCCGAGTTGATGACA3480
TTGTCAGATGGGGAGTTTGGCTGGGGCGGCACATCTGTCAAAAGATAACGCAGGTGTCCT3540
AAGGGGAGCTCATTGAGAACAGAAATCTCAAGTAGAATAAAAGGGTAAAAGTTCCCTTGA3600
TTTTGATTTTCAGTACGAATACAAACCATGAAAGTGTGGCCTATCGATCCTCTAAATCCT3660
CGAAATTTGAGGCTAGGGGTGCCAGAAAAGTTACCACAGGGATAACTGGCTTGTGGCAGC3720
CAAGCGTTCATAGCGACGTTGCTTTTTGATCCTTCGATGTCGGCTCTTCCTATCATACCG3780
AAGCAGAATTCGGTAAGCGTTGGATTGTTCACCCACTAATAGGGAACGTGAGCTGGGTTT3840
AGACCGTCGTGAGACAGGTTAGTTTTACCCTGCTGATGAAGTTATCGCAATGGTAATTCA3900
GCTTAGTACGAGAGGAACCGTTGATTCAGATATTTGGTTTTTGCGGTTGTCTGACCAGGC3960
AGTGCCGCGAAGCTATCATCTGTTGGATTATGGCTGAAAGCCTCTAAGTCAGAATCCATG4020
CCAGAAAGCGATGATATTTCCTCACGTTTTTTGATACAAATAGGCATCTTGCCAATATCA4080
GTATTTGGACGGGTGGAGGCGGACGGAAGTGTTCGTCTCTGTCCATTAATATTAATTAAT4140
ATTCGTGAGGGCGAATCCTTTGTAGACGACTTAGTTGAGGAACGGGGTATTGTAAGCAGT4200
AGAGTAGCCTTGTTGTTACGATCTGCTGAGATTAAGCCTTTGTTCCCAAGATTTGT4256
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 381 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
TCAAAAAGAACATTTCTTCTGAGTGGTGAGGGGTCCGTTAGAGCACACTCGCTCCTTGGA60
AGAGATGTTTTTTTTGATATTAGGAACCAATAGAATATTTAGAATTTAATTTAGATTAAA120
TTATAGAAGGGTATCTGTAGCGATAAGTTTCCATTTCAAATTTTTCTGATGCAGTAGTAT180
GTTCTTTTCTAAAATAAAATGATAGTTTATTAATGATTAAACTAATTATTATCCTTTGGC240
CATCTTTTTCTACATTTTCCAGAAACAGATCTAATTACGTTTTTGCTATCTATAATTATT300
AAAAATAATCATATATCTTTAAAGTTGACCTCAACGTCTTAAAATGTTTAGTTTTTTAAT360
TAACCCTAAACCCTAGAACAC381
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